Bilirubin is a bile pigment which is a metabolic product of heme formed from the degradation of erythrocytes by reticuloendothelial cells. It can also be formed by the breakdown of other heme-containing proteins such as cytochromes. The most typical biological form of bilirubin is bilirubin IXa.
Bilirubin IXa normally circulates in the plasma of the bloodstream in several forms. One form has been designated as "unconjugated" or "indirect" bilirubin. The unconjugated bilirubin may complex to serum albumin and as such is designated as "bound" unconjugated bilirubin, or it may exist in a non-albumin bound or free form and as such it is designated as "unbound" unconjugated bilirubin. Both bound and unbound unconjugated bilirubin are normally transported to the liver, wherein they are taken up by the liver cells and converted to a polar conjugate form. The conversion typically involves the transfer of glucuronic acid molecules, catalyzed by the enzyme hepatic glucuronyl transferase, to the unconjugated bilirubin. This converted bilirubin is designated in the art as "conjugated" or "direct" bilirubin. Some of the conjugated bilirubin may leak back into the bloodstream. Conjugated bilirubin in the bloodstream, like unconjugated bilirubin, can bind to albumin, although the unconjugated form seems to bind to albumin more tightly. Thus, bilirubin in the blood exists in four forms: (1) bound conjugated bilirubin, (2) unbound conjugated bilirubin, (3) bound unconjugated bilirubin and (4) unbound unconjugated bilirubin. To summarize, (1) and (2) together are known as "direct" bilirubin, while (3) and (4) together are termed "indirect" bilirubin. These four fractions generally comprise the serum or plasma bilirubin concentration. However, a fifth component, delta bilirubin, has been described which is a bilirubin covalently linked to albumin. It is a very small fraction of the total bilirubin and not relevant to this discussion.
Unconjugated but not conjugated bilirubin can poison many vital cell functions, and a variety of experimental and clinical evidence suggests that unbound unconjugated bilirubin is a potential neurotoxin. Specifically, since it is not restricted by albumin binding, unbound unconjugated bilirubin can act as a neurotoxin because of its ability to migrate from the vascular space into the nervous system where it can complex with nervous tissue causing irreversible damage. Typically, unbound unconjugated bilirubin comprises less than 0.05% of the fraction of total bilirubin in the blood and is therefore difficult to measure. Disease states resulting in elevated levels of serum bilirubin may raise either "conjugated" or "unconjugated" levels of bilirubin or both forms simultaneously. However, only elevated unconjugated and unbound forms predispose a patient to neurological bilirubin toxicity.
Newborn infants suffering from high levels of unconjugated bilirubin (i.e., hyperbilirubinemia) become jaundiced after birth and are susceptible to developing kernicterus, which is an accumulation of unconjugated bilirubin in tissues of the nervous system, particularly the basal ganglia of the developing brain. This condition, also designated as bilirubin encephalopathy, may produce athetoid cerebral palsy, ocular palsy, deafness, mental retardation, and defects in fine motor control and cognitive function. Neonates afflicted with hemolysis and infants born prematurely compose the highest risk groups for bilirubin encephalopathy; however, kernicterus has also been reported in jaundiced term newborns with no clear pathological etiology for their jaundice.
Most newborns develop transient unconjugated hyperbilirubinemia in the first few days of life. There has been a recent resurgence in bilirubin encephalopathy (kernicterus) in term and near term newborns that has been attributed both to early postnatal hospital discharge and less concern about bilirubin toxicity by health care providers.
Approximately 1-2% (40,000 to 80,000) newborns per year in the United States are readmitted to the hospital for hyperbilirubinemia and about 5% (2000 to 4000 newborn) will have a total bilirubin concentration high enough to consider treatment by exchange transfusion.
Physicians faced with treatment decisions for these hyperbilirubinemic babies must determine whether the jaundice is severe enough to require exchange transfusion. Since both the exchange transfusion and jaundice are associated with significant risks to the patient, including death, the laboratory data on which these decisions are based are very important. The decision to perform exchange transfusion on a newborn is usually based on total conjugated and unconjugated blood bilirubin levels because unbound, unconjugated bilirubin is not easily measured. In fact, no routine clinical laboratory method exists for this purpose. Moreover, the concentration of total bilirubin in the blood has poor sensitivity and specificity in predicting the risk of developing kernicterus. For example, a TBC (total bound bilirubin concentration) of 15 mg/dL when used as the exchange transfusion level for term newborns with hemolysis has a sensitivity of about 83% and a specificity of about 78%. If this criterion were used for medical decision making without additional considerations, about 17% of the babies needing an exchange transfusion would not receive one and would suffer neurological injury. Furthermore, 22% of those not needing an exchange transfusion would receive one anyway along with the attendant risks. Therefore, exchange transfusion treatments may be performed needlessly upon neonates who do not need it while some requiring treatment will not receive it.
Measurement of unbound unconjugated bilirubin (neurotoxic fraction of the blood bilirubin) would be a better way to determine when a jaundiced baby needs an exchange transfusion.
Two classes of medical diagnostic tests have been used to determine the need for treatment by exchange transfusion by measuring levels of substances which are believed to correlate significantly with levels of unbound unconjugated bilirubin in the patient. These tests are based upon the following equation EQU bu+bc+a.revreaction.A:bu,bc
wherein bu is the unbound unconjugated bilirubin, bc is the unbound conjugated bilirubin, a is the serum unbound albumin, and A:bu,bc is the albumin complexed with unconjugated and conjugated bilirubin. Put simply, then the first method measures the unbound albumin (a), while the second method (peroxidase) measures the total amount of unbound unconjugated and unbound conjugated (i.e., bu+bc). More specifically, the first type depends upon measuring circulating levels of albumin binding sites unoccupied by bilirubin (i.e., unbound albumin) to predict the "saturation" of albumin with bilirubin. Since albumin binds with unbound unconjugated bilirubin, it has been suggested that knowledge concerning the level of unbound albumin would correlate indirectly with the amount of unbound unconjugated bilirubin in a patient. This approach assumes that albumin has one primary binding site for bilirubin and that any additional bilirubin binding sites are irrelevant. These tests attempt to determine a total bilirubin concentration at which the albumin will become "saturated" with bilirubin. If the tests determine that the amount of circulating unbound albumin is too small to bind the amount of bilirubin in the blood, exchange transfusion would be recommended. The unbound bilirubin, although not measured directly, is assumed to be proportional to the degree of albumin saturated with bilirubin. Again, when the albumin is deemed saturated with bilirubin, exchange transfusion is recommended.
Such tests, however, have unreliable endpoints and do not accurately reflect the "true" level of unbound unconjugated bilirubin in the patient because a single albumin molecule can bind more than one bilirubin molecule, and the bilirubin molecules may cause allosteric changes in the albumin making the concept of "saturation" ambiguous and the endpoint unreliable.
The second class of tests are not correlative tests, that is, they attempt to measure the actual levels of the various unbound bilirubin species in the blood. This second class of test method is preferred to tests using correlative methods since a test which directly measures the species of bilirubin in a sample typically exhibits a smaller margin of error. However, currently, such tests measure the total amount of unbound species of bilirubin (bc and bu) rather than the neurotoxic fraction of bilirubin in the blood. All bilirubin binding tests described to date do not discriminate between conjugated and unconjugated fraction of the unbound bilirubin.
Clinical laboratories in the U.S. do not routinely measure unbound unconjugated bilirubin concentrations. One non-correlative assay that is currently used in clinical laboratories measures total bilirubin concentration (TBC) in the serum/plasma, measuring the total of all four species of bilirubin (i.e., (1) bound conjugated bilirubin, (2) unbound conjugated bilirubin, (3) bound unconjugated bilirubin, and (4) unbound unconjugated bilirubin). For example, the TBC may be measured using bilirubin's light absorbing properties between 440 and 470 nm. The inherent absorption of bilirubin can be used to measure only the total bilirubin concentration because the differences in the absorption spectra of conjugated and unconjugated bilirubins are too subtle to allow discrimination between the two.
A second non-correlative test that is in clinical use measures both the total bilirubin concentration and the fraction of the total bilirubin that is conjugated. For example, one test measures the serum/plasma total and conjugated (direct) bilirubin concentration by converting bilirubin to a blue-violet colored diazo derivative which absorbs light above 500 nm. In this test, serum or plasma is combined with the diazo reagent (made by combining an organic acid like sulfanilic acid with nitrite at acid pH). Inasmuch as the conjugated bilirubin forms diazo derivatives much faster than unconjugated bilirubin, the direct bilirubin concentration which represents the concentration of conjugated bilirubin in the sample is calculated from the absorbance of light at 565 nm by the initial diazo derivatives. An accelerator such as methanol or caffeine and sodium benzoate is then added which accelerates the reaction of the unconjugated bilirubin with the diazo reagent. The total bilirubin is calculated from the final absorbance at 565 nm of all the diazo derivatives. The difference between the total and conjugated bilirubin is the concentration of unconjugated (indirect) bilirubin. However, even this combined data still does not provide the clinician with sufficient information about the unconjugated unbound bilirubin fraction of a serum plasma sample because it does not distinguish between the two types of unconjugated bilirubin, i.e., that which is bound to albumin and harmless and that which is unbound to albumin and potentially neurotoxic. Yet, it is the unbound unconjugated bilirubin, i.e., a tiny fraction of the TBC, which best assesses the risk of jaundiced newborns for developing bilirubin encapthalopathy. Knowledge of the concentration of unconjugated bilirubin which is unbound to albumin (and therefore potentially neurotoxic) is useful information.
Accordingly, a diagnostic test indicating unconjugated, unbound bilirubin would be preferred since it could specifically and accurately determine the neurotoxic fraction of total bilirubin in the blood. Knowledge of the concentration of unbound unconjugated bilirubin would be advantageous since its concentration may increase exponentially with any linear increase in the concentration of total bilirubin due to the effect of mass action on the binding of bilirubin with albumin. Therefore, an accurate measure of any change in the concentration of unbound, unconjugated bilirubin is desired because this species of bilirubin is extremely relevant in the clinical decision to administer the potential lifesaving but dangerous treatment of exchange transfusion.
A current kinetic technique for noncorrelative measurement of non-albumin bound bilirubin employs the horseradish peroxidase catalyzed oxidation of all species of bilirubin by peroxide (Jacobsen & Wennberg, Clin. Chem. 1974, 20(7), 783-789) (hereinafter referred to as "J & W method"). In this method, horseradish peroxidase catalyzes the oxidation of both conjugated and unconjugated bilirubin by peroxide to form products which are colorless at 460 nm. Bilirubin bound to albumin is protected from oxidation, and only unbound bilirubin is available for oxidation. Since only unbound bilirubin reacts with the peroxide, the reaction velocity of the oxidation of both conjugated and unconjugated bilirubin species is proportional to the concentration of unbound bilirubin within the sample. After measuring the first order rate constant (K.sub.p) for the peroxidase catalyzed oxidation of unconjugated bilirubin by peroxide in albumin free solutions (i.e., where all the bilirubin is unbound), the concentration of unbound bilirubin is determined from the rate of oxidation in the sample. However, the peroxidase test that has been utilized heretofore has several limitations. The sample dilution required (about 40 fold) has been shown to alter binding of bilirubin as well as other ligands (See, for example, Ahlfors, Clin. Chem. 1981, 27, 692-696). In addition, direct (conjugated) bilirubin is non-neurotoxic, but, if present, is also oxidized by the peroxide, causing overestimation of the amount of the toxic unbound bilirubin concentration present in the sample. Moreover, the rate limiting dissociation of bilirubin from its complex with albumin during the test may lower the steady state level of the unbound bilirubin concentration sufficiently during the oxidation to cause significant underestimation of the unbound unconjugated bilirubin in the sample. Therefore, the unbound bilirubin measured by this method does not provide adequate information about the unbound, unconjugated bilirubin, i.e., the neurotoxic fraction (bu).
Therefore, a method which determines the concentration of unbound, unconjugated bilirubin (bu) would be greatly advantageous. The present inventor has developed such a method.